Using his knowledge of how genes are organized and repaired in human cells, Dr. Graham Dellaire, AV俱乐部 Medical School鈥檚 Cameron Research Scientist in Cancer Biology, has developed a technique that could make gene therapy more effective and safer to use. His work was recently published in Nucleic Acids Research and Nature.
CRISPR, named 2015鈥檚 breakthrough discovery of the year, stands for "Clustered Regularly-Interspaced Short Palindromic Repeats." It can accurately target and edit DNA, offering the potential to cure genetic diseases and find new treatments for cancer.
To apply CRISPR in non-dividing cells 鈥� such as those in muscle and brain tissue 鈥� researchers must first make them behave like cells that divide. They do this by turning on a cellular process called homologous recombination, which protects DNA; the recombination allows a cell鈥檚 genes to be manipulated and rearranged without the possibility of causing more harm than good.
鈥淵ou can鈥檛 accurately repair a gene in a non-dividing cell because basic pathways like homologous recombination aren鈥檛 turned on to enable the gene therapy to be carried out,鈥� says Dr. Dellaire, an associate professor and director of research in the Department of Pathology.
鈥淢y colleague, Dr. Daniel Durocher, and his team at Mount Sinai Hospital鈥檚 Lunenfeld-Tanenbaum Research Institute, found a way to re-activate the recombination that鈥檚 needed to do gene editing in a non-diving cell, but the group needed a way of proving it. That鈥檚 where we were able to help.鈥�
Zeroing in on gene targets
In his laboratory at AV俱乐部 Medical School, Dr. Dellaire came up with a fluorescent tagging technique that allows scientists to identify when gene targeting has been successful in a cell 鈥� even in those that don鈥檛 divide.
鈥淥ur technique measures the efficiency of gene editing. We know that we鈥檝e got a precision gene editing event when the cell we鈥檙e working with turns green and a beautiful ring appears around its nucleus,鈥� explains Dr. Dellaire. 鈥淭he green fluorescence then enables us to count tens of thousands of cell samples, so our statistics end up being very good.鈥�
鈥淚t was thought to be impossible to do precision gene editing in a non-dividing cell. And that鈥檚 why Dr. Durocher鈥檚 breakthrough, and our publication in Nature, is so profound. Not only do we have the first evidence that gene editing in non-dividing cells is actually possible, but we鈥檝e got a screening system that will enable us to keep improving gene therapies.鈥�
Up to now, CRISPR has been used to treat diseases that affect parts of the body comprised of cells that divide, such as the blood.
This new knowledge will enable gene therapy to be applied to disorders of the musculoskeletal and nervous systems 鈥� systems that are made up of non-diving cells. It could also be used to fight certain types of infections.
鈥淚f you have a life-long, latent infection with a virus such as herpes, we could use CRISPR gene therapy to get rid of it,鈥� says Dr. Dellaire. 鈥淚magine if an ingredient in your cream could delete the genome of the herpes virus, and you never get a blister on your lip again.鈥�
Mining for molecules
鈥淚n order to get non-dividing cells to behave like dividing ones, Dr. Durocher had to knock out important DNA repair genes,鈥� explains Dr. Dellaire. 鈥淪ince that can only be done in the lab and not in people, we鈥檙e now trying to identify molecules that will take the place of these man-made genetic manipulations. Our hope is that that we can find molecules that will one day make gene therapy a reality for diseases like muscular dystrophy.鈥�
Looking for these molecules is an active area of research by biotech companies around the world.
鈥淥ur fluorescent tagging system is so unique and so efficient that we have a chance at finding molecules that these companies may not find. It鈥檚 kind of like a gold rush at this point, and it鈥檚 very important to dig in the right spot,鈥� says Dr. Dellaire.
Partnering with the Vancouver-based Centre for Drug Research and Development (CDRD), Dr. Dellaire is working on identifying molecules that can be commercialized and developed into drugs. Currently funded by the Canadian Institutes of Health Research, he鈥檚 also looking for more research money.
鈥淲e could be world leaders in this area,鈥� says. Dr. Dellaire. 鈥淭he CDRD is ready to start screening. We just need the funding.鈥�
Making gene therapy safer
More than 20 years ago, children born with a genetic disorder called severe combined immunodeficiency were the first people to be treated with gene therapy. It led to leukemia.
鈥淭hese kids were being cured with a viral gene remedy. A virus was used to replace their faulty genes by inserting a new, healthy gene into their DNA. This just captivated the world; it was front-page news,鈥� remembers Dr. Dellaire. 鈥淏ut by inserting the virus randomly into the genome and changing the children鈥檚 DNA, mutations formed.鈥�
While CRISPR鈥檚 still not efficient enough to be better than using viruses for gene therapy, it鈥檚 getting there.
Using his fluorescent screening technique, Dr. Dellaire found that some molecules are capable of enhancing gene targeting by up to six-fold. However, they鈥檙e potentially too toxic for use in patients, so there鈥檚 a need to find new ones.
鈥淏ecause there鈥檚 a danger in using viruses to insert genes into our DNA, we hope our screening system will help identify molecules that will enhance CRISPR鈥檚 effectiveness,鈥� says Dr. Dellaire. 鈥淭he ultimate goal would be to replace viral gene therapies altogether, making gene therapy more efficient, more effective, and safer for patients.鈥�